Peptide sequencing directly from solid surfaces

Abstract

Method and system for sequence analysis directly on a solid surface that is both high speed and high throughput are described herein, utilizing equipment available in most protein analysis facilities. For example, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a concatenated set of charged peptide fragments that differ by a single amino acid. The fragments are taken up in a small volume of matrix solution and analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide sequences can be read directly from the resulting spectra.

What is claimed is: 
     
         1 . A method for sequencing a peptide, comprising:
 derivatizing the peptide containing N peptide bonds;   cleaving the derivatized peptide into a set of peptide fragments, wherein the set of peptide fragments are generated by cleaving at each of the N peptide bonds;   sequencing the set of peptide fragments to yield a peptide sequence of said peptide.   
     
     
         2 . The method of  claim 1 , wherein the peptide is coupled to a solid surface. 
     
     
         3 . The method of  claim 2 , wherein the peptide is synthesized in situ on the solid surface. 
     
     
         4 . The method of  claim 2 , wherein the peptide is obtained from protein digestion and attached to the solid surface. 
     
     
         5 . The method of  claim 2 , wherein the solid surface is a resin bead. 
     
     
         6 . The method of  claim 2 , wherein the solid surface is a peptide microarray, wherein the microarray comprises a glass or on a silicon wafer. 
     
     
         7 . The method of  claim 1 , wherein the set of peptide fragments comprises various lengths. 
     
     
         8 . The method of  claim 1 , wherein the derivatizing step comprises attaching a positive charge-bearing group to a N-terminus of a peptide. 
     
     
         9 . The method of  claim 8 , wherein the positive charge-bearing group is N-Tris(2,4,6-trimethoxypheyl)phosphonium acetic acid (TMPP). 
     
     
         10 . The method of  claim 8 , wherein the positive charge-bearing group is trialkylammonium acetyl (TAA). 
     
     
         11 . The method of  claim 8 , wherein the attaching a positive charge-bearing group to a N-terminus of a peptide step is performed prior to a deprotection step of the peptide. 
     
     
         12 . The method of  claim 8 , wherein the attaching a positive charge-bearing group to a N-terminus of a peptide step is performed subsequently to the deprotection of a the peptide. 
     
     
         13 . The method of  claim 11  or  12 , wherein the deprotection step comprises removing a protecting group from an amino acid. 
     
     
         14 . The method of  claim 1 , wherein cleaving step comprises treating the peptide in ammonia gas for a length of time. 
     
     
         15 . The method of  claim 14 , wherein the ammonia gas has a pressure ranging from about 15 psi to about 100 psi. 
     
     
         16 . The method of  claim 14 , wherein the length of time is about 1 hour to about 5 hours. 
     
     
         17 . The method of  claim 1 , wherein the sequencing step further comprising extracting the set of peptide fragments by a matrix solution and analyzing the set of peptide fragments by a mass spectrometry. 
     
     
         18 . The method of  claim 17 , wherein the mass spectrometry is a matrix assisted laser desorption/ionization (MALDI) mass spectrometry. 
     
     
         19 . A system for sequencing a peptide directly from a solid surface, comprising:
 a container for derivatizing the peptide containing N peptide bonds;   a chamber containing ammonia gas for cleaving the derivatized peptide into a set of peptide fragments, wherein the set of peptide fragments are generated by cleaving at each of the N peptide bonds; and   a mass spectrometer for sequencing the set of peptide fragments to yield a peptide sequence of said peptide.   
     
     
         20 . The system of  claim 19 , wherein the solid surface is a resin bead or a peptide microarray.